Tumor growth and metastasis impact a large number of people each year. It is estimated that over 600,000 new cases of cancer will be diagnosed in the United States per year (Varner, J., et al., Cell Adh. Commun. 1995; 3:367-374).
Metastasis, the spread of malignant tumor cells from the primary tumor mass to distant sites involves a complex series of interconnected events. (Liotta, et al., Cell 1991; 64:327-336; Wyckoff, et al., Cancer Res. 2000; 60:2504-2511; Kurschat, et al., Clinc. Exp. Dermatol. 2000; 25:482-489.) The metastatic cascade is initiated by a series of genetic alterations leading to changes in cell-cell interaction, which allow tumor cells to dissociate from the primary tumor mass. The dissociated cells locally invade and migrate through proteolytically modified extracellular matrix (ECM). The dissociated cells gain access to the circulatory system. To establish a metastatic deposit, the circulating tumor cells must evade host immune defenses, arrest in the microvasculature, and extravasate out of the circulation. The tumor cells then invade the ECM at the new site, proliferate, induce angiogenesis, and continue to grow.
Therapies designed to block angiogenesis may significantly effect the growth of solid tumors and metastases. Blocking tumor neovascularization significantly inhibits tumor growth in various animal models, and human clinical data is beginning to support this contention as well (Varner, J., et al., Cell Adh. Commun. 1995; 3:367-374). These and other studies suggest that the growth of solid tumors requires new blood vessel growth for continued expansion of the tumors beyond a minimal size (Varner et al., 1995; Blood, C. H., et. al., Biochim. Biophys. Acta. 1990; 1032:89-118; Weidner, N. et al. J Natl. Cancer Inst. 1992; 84:1875-1887; Weidner, N. et al., N. Engl. J Med. 1991; 324:1-7; Brooks, P. C. et al. J Clin. Invest. 1995; 96:1815-1822; Brooks, P. C. et al., Cell 1994; 79:1157-1164; Brooks, P. C. et al. Cell 1996; 85:683-693; Brooks, P. C. et al., Cell 1998; 92:391-400). Inhibition of angiogenesis is, therefore, a promising treatment for cancer and metastatic disease.
Angiogenesis is the physiological process by which new blood vessels develop from pre-existing vessels (Varner et al., 1995; Blood et al., 1990; Weidner et al., 1992). This complex process requires cooperation of a variety of molecules including growth factors, cell adhesion receptors, matrix degrading enzymes and extracellular matrix components (Varner et al., 1995; Blood et al., 1990; Weidner et al., 1992).
Inhibition of angiogenesis may also be useful in treating other diseases that are characterized by unregulated blood vessel development including, for example, ocular diseases (e.g., macular degeneration and diabetic retinopathy) and inflammatory diseases (e.g., arthritis and psoriasis) (Varner et al., 1995).
Many investigators have focused their anti-angiogenic approaches towards growth factors and cytokines that initiate angiogenesis (Varner et al., 1995; Blood et al., 1990; Weidner et al., 1992; Weidner et al., 1991; Brooks et al., 1995; Brooks et al., 1994; Brooks et al., 1997). There are, however, a large number of growth factors and cytokines that have the capacity to stimulate angiogenesis. The therapeutic benefit of blocking a single cytokine, therefore, may have only limited benefit due to this redundancy. Little attention has been directed to other anti-angiogenic targets.
Recent studies have suggested that angiogenesis requires proteolytic remodeling of the extracellular matrix (ECM) surrounding blood vessels in order to provide a microenvironment conducive to new blood vessel development (Varner et al. (1995); Blood et al. (1990); Weidner et al. (1992); Weidner et al. (1991); Brooks et al. (1995); Brooks et al. (1994); Brooks et al. (1997)). The extracellular matrix protein collagen makes up over 25% of the total protein mass in animals and the majority of protein within the ECM.
Inhibition of angiogenesis would be a useful therapy for restricting tumor growth and metastases. Inhibition of angiogenesis may be effected by (1) inhibition of release of “angiogenic molecules” such as, for example, bFGF (basic fibroblast growth factor), (2) neutralization of angiogenic molecules, (e.g., anti-bFGF antibodies), and (3) inhibition of endothelial cell response to angiogenic stimuli. (Folkman et al., Cancer Biology, 3:89-96 (1992)). Several potential endothelial cell response inhibitors have been described that might be used to inhibit angiogenesis, e.g., collagenase inhibitors, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D3 analogs, and alpha-interferon. Additional proposed inhibitors of angiogenesis have also been described in the literature. (Blood, et al. (1990); Moses et al. (1990) Science 248:1408-1410; Ingber, et al. (1988) Lab. Invest., 59:44-5 1; and U.S. Pat. Nos. 5,092,885; 5,112,946; 5,192,744; and 5,202,352.)
Collagen is an extracellular matrix protein containing a [Gly-Xaa-Xaal]n sequence motif. Collagen types are well known in the art (see, e.g., Olsen, B. R. (1995) Curr. Op. Cell. Biol. 5:720-727; Kucharz, E. J. The Collagens: Biochemistry and Pathophysiology. Springer-Verlag, Berlin, 1992; Kunn, K. in Structure and Function of Collagen Types, eds. R. Mayne and R. E. Burgeson, Academic Press, Orlando). Collagen is a fibrous multi-chain triple helical protein that exists in numerous forms (Olsen, B. R. (1995) Curr. Opin. Cell Biol 7, 720-727; Van der Rest, M., et al. (1991) FASEB 5, 2814-2823). At least 18 genetically distinct types of collagen have been identified, many of which have distinct tissue distributions and functions (Olsen (1995); Van der Rest, et al. (1991)). Collagen type-I is the most abundant collagen in the extracellular matrix. Collagen type-I, collagen type-III, collagen type-IV and collagen type-V have been shown to be associated with all pre-existing blood vessels in vivo.
The mature collagen molecule is composed of two α1 chains and one α2 chain twisted into a triple helix. Collagens type-I and type-IV, for example, are composed of major chains designated α1(I) and α2(I) and α1(IV) and α2(IV), respectively. In vivo, collagen is normally found in the mature triple helical form.
Denaturation of the native three dimensional structure of mature triple helical collagen may expose cryptic regulatory regions that control angiogenesis. Disruption by antibodies of cellular interactions with denatured collagen type-IV blocks tumor growth and angiogenesis (Xu, J., et al. (2001) J. Cell Biol. Vol. 154:1069-1079; Hangia, et al. (2002) Am. J. Pathol. Vol. 161:1429-1437). Brooks et al. (PCT WO 00/40597) discloses antibodies that bind to cryptic regions within various denatured collagen types.
It has now been surprisingly discovered that peptide antagonists selective for denatured collagen type-IV inhibit angiogenesis and tumor growth. Peptide antagonists that specifically bind to denatured collagen type-IV provide the basis for powerful new compounds for treating cancer, inflammatory diseases and other angiogenesis-associated diseases.